Human Amniotic Membrane vs Collagen in the Treatment of Superficial Second-degree Burns in Children

Scald burns in children are a common presentation to the emergency department (ED). Light et al¹ highlight several challenges in the management of burns in developing countries, including larger burn size, longer contact times, baseline malnutrition and anemia, lack of awareness of first aid, delayed presentation, refusal of care because of limited finances, and inability to access health care. Burn injury also has devastating long-term physical, psychological, and financial consequences for the child and family. The ideal dressing would require a 1-time application in the ED and would relieve pain, be readily available, be inexpensive, expedite healing, and reduce scarring.

Human amniotic membrane (HAM) is a byproduct of pregnancy that is typically discarded after birth. It has been used as a biologic wound dressing for centuries, and in 1913, Sabella² was the first to report its use in the management of burns. Human amniotic membrane contains fibronectin, proteoglycans, glycosaminoglycans, laminins, and type IV, V, and VII collagen, as well as fibroblasts and pluripotent stem cells, all of which are required for tissue growth and repair.³ It also contains interleukin-4, -6, -8, and -10, and it is hypothesized that the immunosuppressive functions of these interleukins contribute to the immune-privileged properties of the amniotic membrane.³ These biologic characteristics are thought to contribute to the clinical efficacy of HAM in terms of reduced pain and infection, faster healing, and decreased scarring.^3-5 Harvesting HAM has been described as labor intensive and time consuming, however.⁵ A simplified method of harvesting HAM is described herein.

Commercially available collagen, which resembles HAM, is made from reconstituted bovine collagen (Xenoderm WT; Helix Pharma). This collagen is a thin, transparent, acellular sterile sheet that is devoid of growth factors and stem cells. Both materials have been used as the inner layer of burn dressings.^3-7

This study describes a 1-time application of collagen and HAM that can dry naturally without the need to cover the application with additional dressing material. After these materials are completely dry, they form a hard, shell-like layer (hereafter referred to as exoskeleton); this process takes approximately 6 to 8 hours. The child is pain free once the exoskeleton is formed, and the exoskeleton can be touched without causing pain. However, the exoskeleton must be kept dry until reepithelialization takes place beneath it and the shell falls off or is shed. The purpose of this study was to determine whether HAM and collagen used in this manner are viable dressing options in children with second-degree burns by assessing percentage of the dressing take, need for reapplication, wound infection, time to wound healing, and scar quality.

Patients
A single-blind (outcome assessment), internally randomized clinical trial was conducted in the Department of Pediatric Surgery at Christian Medical College (Vellore, Tamil Nadu, India), which is a tertiary hospital in India that serves 1 million pediatric outpatients each year.⁸ The study was funded by an institutional research grant and was conducted over 14 months (Clinical Trials Registry of India [CTRI/2015/07/005974]), with a convenience sample of patients enrolled to coincide with the availability of the primary investigator (A.R.D.).

Children aged 16 years and younger with superficial second-degree scald burns affecting less than 20% of the body surface area and who presented within 24 hours of the incident were included. Children with only epidermal burns, full-thickness burns, burns involving 20% or more of the body surface area, or signs of systemic sepsis at presentation, or whose parents refused to participate in the study were excluded.

The primary outcomes were percentage take of the dressing, need for reapplication in areas of dressing loss, wound infection, time to wound healing, and scarring. Explanatory variables included age; sex; details of the incident, including mode, time, and place of burn; first aid administered; percentage body surface area involved; time to arrival to the hospital; and time to dressing application.

Human amniotic membrane ­preparation
Fresh placenta obtained from elective cesarean section deliveries with negative viral and venereal serology and prior informed consent was used to make HAM in-house at the Christian Medical College, Vellore. The amniotic membrane was harvested as soon as the baby was expelled, when there was the least amount of blood contamination, thereby saving time that otherwise would have been spent removing blood clots and debris from the membrane. The membrane was easily peeled away from the chorion and was washed to produce a translucent membrane. It was kept in a sterile 100-mL glass bottle containing gentamicin and crystalline penicillin in normal saline for a maximum of 1 week at a temperature of 4°C.

Application and internal ­randomization
Informed consent was obtained from the patient or guardian. Collagen and HAM were applied following the administration of appropriate analgesia, with or without sedation. The burn wound was cleaned with normal saline and photographed. The dressing was rinsed in sterile normal saline before being applied aseptically. An approximately equal part of each dressing, randomly assigned, was applied to each burn area. Figure 1 shows the appearance of the dressings immediately after application. Because the dressings were indistinguishable, on each photograph the application area was labeled with an X for collagen or a Y for HAM. The wounds were left uncovered after application, and the membranes dried to form a hard, painless exoskeleton. Regular simple oral analgesics were prescribed. The only aftercare required was the need to keep the area dry until the exoskeleton was shed.

Outcome assessment
Alternate day clinical examination and photography were used to monitor the children for dressing uptake, pain, itching, and any local infection or evidence of wound exudate collection. The day the dressing shed with epithelialization beneath it was used to calculate the time to wound healing. Scars were evaluated using photographs taken 3 and 6 months after the incident. All photographs were taken with the same camera in natural sunlight in a designated location. On each photograph the areas treated with collagen and HAM were digitally labeled with an X and a Y, respectively, and saved. At the end of the study, the scars were evaluated by 2 independent burn surgeons blinded to the material used. The Vancouver Scar Scale (VSS) was used to score each half of the scar separately. The maximum possible score of VSS is 13 and a score of 0 is normal.

Cost analysis
The cost of preparing HAM was calculated based on 2 hours of hospital staff labor as well as the costs of cleaning and storing. A 25-cm × 25-cm sheet (the approximate product of 1 placenta) was estimated to cost 300 Indian Rupee (INR). In contrast, at the time of this writing the cost of a 10-cm × 25-cm sheet of collagen is 740 INR.

Ethics
The study was conducted in compliance with the ethical rules for human experimentation stated in the Declaration of Helsinki, 1975 revision. Institutional review board (IRB) and ethical clearance were obtained before the study commenced (IRB number 9310 dated 9.3.2015). The study was registered under the Clinical Trials Registry of India (CTRI/2015/07/005974). The Institutional Data Safety and Monitoring Board periodically monitored the study outcomes.

Statistical analysis
Statistical analysis was done using SPSS (version 16.0; IBM) and diagnostic test software developed at Christian Medical College’s Department of Biostatistics. Descriptive statistics were used for demographic variables, including percentages, median, and interquartile range (IQR). The weighted Cohen kappa statistic was used to analyze the results of the VSS. The scars were categorized into group A (≤3 points) and group B (>3 points) based on the VSS scores. Continuous variables were analyzed using the Mann-Whitney test. Survival analysis was done using the Kaplan-Meier method.

A total of 43 children (25 boys, 18 girls) were enrolled in the study. The scalding agent was water in 23 children, milk or tea in 12, soup or rice water in 6, and oil in 2. Detailed demographic information is noted in the Table.

None of the children received first aid in the form of cold running water for 20 minutes immediately after the burn occurred. Before arriving at the hospital, 25 children (58%) were treated at private clinics or with traditional medications such as silver sulfadiazine or other medicated ointment, gentian violet, wheat flour, and banana juice.

A total of 25 children (58%) were treated as outpatients, and 18 (42%) required hospital admission. Reasons for admission included age younger than 1 year, involvement of high-risk areas (face, perineum), and the need to facilitate follow-up (eg, inconvenient travel time or mode of transport). The median length of stay for children admitted to the hospital was 2 days (IQR, 1.5–3 days).

A successful 1-time application was recorded in 40 children (93%). The parents agreed that their child was pain free after the dressing dried and allowed handling of the exoskeleton over the burned area. Sixteen children (37%) had a low-grade fever at the time of application and were treated with simple antipyretic agents. Antibiotics were not required. Eight of the 40 children (20%) who underwent successful applications reported itching at the dressing site, but whether this was due to HAM or collagen could not be determined. A typical dressing application, healing, and scarring pattern are shown in Figure 2.

The dressing failed to take or was removed in 3 children (7%). A child with burns covering 13% total body surface area involving the buttocks and back had issues with soiling, which necessitated removal of the dressing. The cultures grew Klebsiella and Pseudomonas. This case was reported as a serious adverse event to the Institutional Data Safety and Monitoring Board. The wound was managed with silver sulfadiazine dressings and did not require grafting. Two children had poor primary uptake and accumulation of serous fluid beneath the membranes. One child presented with scald burns caused by water over 9% of the chest and abdominal wall. Silver sulfadiazine had been applied to the wound before presentation to the hospital. The other child presented with scald burn caused by milk over the forearm and abdomen; that child had received no prior treatment.

Small pockets of fluid accumulated under the dressing in 8 children (18%). These were managed with needle aspiration or stab incisions in 5 children (12%). Small membrane areas were cut out in 3 children (7%), and triple-antibiotic ointment was applied. These areas continued to heal, but it took longer for the membrane to exfoliate in the collection areas.

In 53% of patients, the HAM and collagen shed together, whereas in 31% HAM peeled away first and in 16% collagen peeled away first. The median time to healing for both HAM and collagen was 10 days (IQR, 8–12 days). The Kaplan-Meier curve shown in Figure 3 depicts the graphical comparison of the time to healing. Healing times by causative agents are depicted in Figure 4. Scalds due to water healed faster than those caused by other liquids, especially hot oil.

Three children were not available for 3- and 6-month follow-up after completion of the initial treatment and were excluded from endpoint analysis. For the remaining 40 patients, the total VSS score ranged from 0 to 10, with a cumulative median of 1 for HAM and collagen. Figure 5 shows the outcome at 3 months and 6 months in 1 child. Based on VSS score, there was no significant difference in scarring between the materials studied in all 40 children. The interrater reliability of the VSS was 89% (X = 45.3% [95% CI, 0.261–0.645]; Y = 51.2% [95% CI, 0.296–0.728]).

Longer healing times were associated with a higher (worse) VSS score. The median healing time was 10 days in group A (VSS ≤ 3) and 12 days in group B (VSS > 3). This difference was statistically significant (P <.001).

A wound swab was taken prior to dressing application in 22 children with clinical suspicion of infection or who received first aid with organic substances. Eight of these cultures were sterile, 8 grew skin contaminants, and 6 had significant bacterial growth. Three of the 6 children with significant bacterial growth experienced clinical wound infection and received antibiotics. Membranes were removed entirely in 1 patient, and the other 2 patients required fluid aspiration or partial removal of ­membranes.

Outpatient treatment of children with minor burns is ideal and achieves the goal of value-based care, which is particularly important in developing countries. Newer topical treatments are making such treatment possible. This study investigated a novel application of 2 readily available materials for use as a 1-time ambulatory dressing option. Both materials were appropriate for this type of application, each acting as a barrier dressing that did not need to be changed. Both dried in 6 to 8 hours at room temperature. Children were pain free after formation of the exoskeleton, and the exoskeleton could be touched without causing pain. To avoid cracking of the dressing at the joints and difficulty with joint mobilization, it was necessary to apply pieces separately on either side of joints. Neither ongoing sedation nor narcotic analgesia was required. As a result, children were able to feed without interruption. This is especially important in developing countries, in which children are frequently malnourished at the time of presentation. The study authors found a lack of awareness of first aid among the study population. None of the children received first aid in the form of cold running water for 20 minutes immediately after the burn occurred. Instead, several home remedies and traditional applications were tried. A concerted effort by the government of India to educate the public about first aid through electronic and social media may make a difference.

Complete dressing take was achieved in 93 children (40%). In 37% of these children (16 of 43), initial low-grade fever was managed with simple antipyretic agents, and antibiotics were not required. Low-grade fever may be attributed to the inflammatory response to the burn injury.⁹ The itching experienced by 20% of patients in this study is a known marker of epithelialization. Itching can be annoying, but it was not reported to be severe or ongoing. In a meta-analysis of randomized controlled trials on the efficacy and feasibility of the use of HAM for burns, Yang et al⁵ reported that compared with conventional methods, HAM produced better reepithelialization and less invasive bacterial colonization. In the experience of the authors of the current study, the 2 dressing materials were comparable in terms of ease of application, need for reapplication, time to healing, and scarring. Human amniotic membrane is less expensive than collagen and thus, HAM is ideal for use in situations in which finances are constrained or in remote areas where collagen may not be available. Immediate harvest of HAM at delivery reduces the time and effort required for its preparation. Stringent measures must be taken to ensure sterility in preparation and minimize the risk of disease transmission.

This novel application technique presented some challenges. In 8 children, blebs of serous fluid accumulated under the membranes. This problem was solved by creating tiny cuts in the material to allow the fluid to extrude or for aspiration. However, doing so defeats the purpose of the barrier dressing. Subsequently, the authors applied antibiotic powder on the surface to facilitate absorption and drying with some success. In select patients with excess exudate, an initial closed method with the application of absorbent bandages over the biologic dressing and subsequent trial of drying after 24 hours was tried. Application in the perianal region was plagued by fecal soiling. For children who require application in this anatomic region, it is best to treat them as inpatients with a fecal management system (eg, Foley catheter, rectal tubing, or commercially available device) and closed dressing to avoid contamination.¹⁰ This is an ongoing unresolved issue in most burn centers in which children are treated.¹⁰

The use of some organic materials (eg, banana juice) as first aid may result in infection. Despite this, only 3 of the 43 children had clinical wound infections, with 1 infection caused by fecal contamination. Infection rates in the patient cohort in this study were significantly lower than average in the same demographic (6% vs 36%).¹¹ The average time to wound healing for both groups of patients in the current study was 10 days. These healing times are shorter than those reported in other studies with the same demographic, as well as the authors’ previous experience of 15 ± 2 days.^1,11 Moreover, most children could be treated as outpatients.

In this study, wound depth was determined clinically because laser Doppler imaging is not available at the authors’ institutions. Although laser Doppler imaging is ideal for determining wound depth, it requires general anesthesia. In most burns, the wound depth is not uniform. Typically, wound depth is greatest at the point of first contact. Thus, a single wound may have varying depths, resulting in different healing times and producing mixed scarring patterns. As a result, whether a wound is covered with HAM or collagen, the scar in an area that sustained a deeper burn would have a higher (ie, worse) VSS score. In this study, however, the dressing was randomly assigned and the evaluation was consistent. As a result, the authors think that variation in wound depth has not affected overall results. Furthermore, another limitation is that scars were assessed using the VSS. Use of ultrasonography and a device to measure skin firmness and elasticity would provide an objective measurement and further strengthen the study results. Interobserver variation in assessment of scar using the VSS was low.

A split wound model was chosen for dressing application to eliminate interpatient confounding factors such as nutritional status, anemia, burn type and time to treatment, infection, hygiene, and living conditions, which can vary markedly in the population served by the authors. As a result, the authors could neither determine whether itching was caused by amnion or collagen nor conduct a detailed cost analysis.

Human amniotic membrane and collagen are safe and afford a 1-time ambulatory option for burn dressing. Time to complete wound healing and scarring are comparable between the 2 treatment options. Earlier shedding of the dressing is predictive of a better scar. Human amniotic membrane is inexpensive and simple to prepare and store. It is an excellent choice for use in economically disadvantaged areas in which collagen may not be available.